Signal Transducer and Activator of Transcription 5 (STAT5) is an inducible transcription factor that plays a pivotal role in the progression of many cancers, including acute and chronic myeloid leukemia (AML, CML). Kinase oncoproteins activated by human leukemia-relevant genetic alterations signal to activate STAT5, and genetic experiments have shown that STAT5 is a requisite downstream effector of these oncoproteins in a major fraction of adult leukemia. While the genetic data are compelling, genetic deletion is not a functional therapy in humans. Proving that STAT5 is a target for the treatment of leukemia requires the use of chemical STAT5 inhibitors in vivo. Drugs targeting upstream STAT5-activating kinases met with initial success, but are plagued with acquired resistance, off-target toxicity associated with poor kinase selectivity, and alternative oncogenic pathways activating STAT5. Moreover, they do not directly inhibit STAT5. A central concept underlying this proposal is that small molecule binding to the STAT5-SH2 domain inhibits STAT5 activation, resulting in a blockade to STAT5 activity. We hypothesize that interrogating our STAT5-SH2 domain inhibitor platform using an integrated medicinal chemistry and leukemia modeling approach will derive the first STAT5- targeted small molecule inhibitor for the in vivo dissection of cancer biology. We expect to validate STAT5 as a bona fide target for the treatment of leukemia, and to provide succinct STAT5-regulated biomarkers to evaluate STAT5-inhibitor efficacy in the context of human AML.